DESCRIPTION (provided by applicant: The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that infects an exceptionally broad range of host cell types. The mechanisms that regulate the broad host specificity of P. aeruginosa host remain largely unknown. Previous studies of how P. aeruginosa regulates expression of its virulence factors have largely focused on chemical cues such as quorum sensing and nutrient availability. The goal of my work is to investigate whether mechanical cues regulate P. aeruginosa virulence and colonization. During the infection process, bacteria encounter a variety of mechanical forces such as adhesion forces when bacteria attach to host cells and directional forces in liquid environments. Detecting mechanical cues in host organisms could thus be part of an infection strategy. My preliminary data show that P. aeruginosa activates virulence genes as cells transition from swimming to adhesion on abiotic surfaces. In addition, my previous work shows that P. aeruginosa responds to the mechanical effects of fluid flow with striking changes in surface motility and surface adhesion, which are determining factors in host colonization. Based on these findings, I hypothesize that specific mechanical cues activate signal transduction pathways that regulate colonization and virulence in P. aeruginosa cells. In this study, I will characterize how mechanical stimuli regulate virulence and colonization using an interdisciplinary approach that combines molecular biology, cell biology, mechanical engineering, and physics. I have enlisted guidance from Dr. Howard Stone, who has expertise in fluid dynamics, Dr. Joshua Shaevitz, who has expertise in bacterial biophysics, and Dr. George O'Toole, who has expertise in P. aeruginosa virulence and biofilm formation. I will test the hypothesis that P. aeruginosa virulence is activated by mechanical cues during the transition from swimming to adhesion on mammalian host cell surfaces. Using optical tweezers, microfluidics, and atomic force microscopy, I will test whether direct mechanical stimulation of cells is sufficient to activate virulence. I will also characterize the P. aeruginosa transcriptionl response to mechanical stimulation by fluid flow and the effect of fluid flow on colonization of P. aeruginosa cells on host cells surfaces. Finally, I will test the hypothesis that the proteins PilX and PilY1 are the mechanosensors that regulate virulence and colonization. Altogether, these experiments will determine the role of mechanical forces in the bacterial infection process. These insights will provide a foundation for developing novel approaches to antibiotic therapies that perturb the ability of P. aeruginosa to infect a broad range of host organisms. In addition, this study would represent one of the first attempts to characterize mechanosensation as a regulator of virulence and colonization in bacteria.